In mammals, the development of spermatozoa from spermatids (spermiogenesis) is characterized the displacement of histones, at which time two major basic transition nuclear proteins (TPI and TP2) become predominant in the nucleus. These are in turn replaced by protamines. The long-term objective is to determine the roles of these transition proteins in the formation of a spermatozoon that is capable of efficient fertilization and transmission of intact genetic material. The roles of the transition proteins will be studied using mice with knockout mutations of the genes Tnpl and Tnp2, for TP1 and TP2, respectively. The hypothesis to be tested is that the transition proteins have an important role in spermatid development and formation of a functional sperm, but their role is indirect through mediating the orderly transitions of nuclear proteins. Specific Aim I will examine spermatid development including the timing and completeness of histone removal in the Tnp-null mutants, the equality of sharing of Tnp-gene products in heterozygotes, the mechanisms of compensation for the transition protein that was eliminated, and the mechanism of loss of spermatids when all transition protein genes are eliminated. Specific Aim 2 will focus on the defects in chromatin structure observed in Tnp-null mutants and the mechanisms by which they arise. Ultrastructural, immunocytochemical, biochemical, and DNA damage analysis techniques will be employed to study the process of chromatin condensation in spermatids and the resulting degree of condensation in spermatozoa. The role of the persistence of incompletely processed protamine 2, which is synthesized as a precursor protein and is incompletely processed in Tnp-mutants, in the incomplete condensation of the sperm, the apparent strand breakage in the DNA, and infertility will be elucidated. Specific Aim 3 will study the relationship between the sperm biochemical and morphological abnormalities in Tnp-mutants and the resulting defects in sperm function, such as mobility, ability to bind to and fertilize and egg, and post-fertilization events of decondensation and development. The phenotypes of the subfertile Tnp-mutant mice are similar to human males with low fertility, not only in general aspects such as defects in morphology and motility, but in presence of strand breakage and incomplete processing of protamine 2 and incomplete chromatin condensation. Thus they provide a well-defined model system for study of human infertility.